Applied Microbiology and Biotechnology

, Volume 66, Issue 3, pp 285–290 | Cite as

Carbon isotope fractionation during cistrans isomerization of unsaturated fatty acids in Pseudomonas putida

  • Hermann J. Heipieper
  • Grit Neumann
  • Nadja Kabelitz
  • Matthias Kastner
  • Hans Hermann Richnow
Biotechnologically Relevant Enzymes and Proteins


The molecular mechanism of the unique cis to trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12 was studied. For this purpose, the carbon isotope fractionation of the cistrans isomerase was estimated. In resting cell experiments, addition of 3-nitrotoluene for activation of the cistrans isomerase resulted in the conversion of the cis-unsaturated fatty acids into the corresponding trans isomers. For the conversion of C16:1 cis to its corresponding trans isomer, a significant fractionation was measured. The intensity of this fractionation strongly depended on the rate of cistrans isomerization and the added concentration of 3-nitrotoluene, respectively. The presence of a significant fractionation provides additional indication for a transition from the sp2 carbon linkage of the cis-double bond to an intermediate sp3 within an enzyme–substrate complex. The sp2 linkage is reconstituted after rotation to the trans configuration has occurred. As cytochrome c plays a major role in the catabolism of Cti polypeptide, these findings favour a mechanism for the enzyme in which electrophilic iron (Fe3+), provided by a heme domain, removes an electron of the cis double bond thereby transferring the sp2 linkage into sp3.


  1. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917Google Scholar
  2. Chatgilialoglu C, Ferreri C, Ballestri M, Mulazzani QG, Landi L (2000) cistrans isomerization of monounsaturated fatty acid residues in phospholipids by thiyl radicals. J Am Chem Soc 122:4593–4601CrossRefGoogle Scholar
  3. Chatgilialoglu C, Zambonin L, Altieri A, Ferreri C, Mulazzani QG, Landi L (2002) Geometrical isomerism of monounsaturated fatty acids: thiyl radical catalysis and influence of antioxidant vitamins. Free Radic Biol Med 33:1681–1692CrossRefPubMedGoogle Scholar
  4. Diefenbach R, Keweloh H (1994) Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids. Arch Microbiol 162:120–125CrossRefPubMedGoogle Scholar
  5. Ferreri C, Costantino C, Landi L, Mulazzani QG, Chatgilialoglu C (1999) The thiyl radical-mediated isomerization of cis-monounsaturated fatty acid residues in phospholipids: a novel path of membrane damage? Chem Commun 5:407–408Google Scholar
  6. Guan XM, Fisher MB, Lang DH, Zheng YM, Koop DR, Rettie AE (1998) Cytochrome P450-dependent desaturation of lauric acid: isoform selectivity and mechanism of formation of 11-dodecanoic acid. Chem Biol Interact 110:103–121CrossRefPubMedGoogle Scholar
  7. Hartmans S, Smits JP, van der Werf MJ, Volkering F, de Bont JAM (1989) Metabolism of styrene oxide and 2-phenylethanol in the styrene-degrading Xanthobacter strain 124X. Appl Environ Microbiol 55:2850–2855Google Scholar
  8. Hartmans S, van der Werf MJ, de Bont JAM (1990) Bacterial degradation of styrene involving a novel flavin adenin dinucleotide-dependent styrene monooxygenase. Appl Environ Microbiol 56:1347–1351Google Scholar
  9. Heipieper HJ, de Bont JAM (1994) Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of the fatty acid composition of membranes. Appl Environ Microbiol 60:4440–4444PubMedGoogle Scholar
  10. Heipieper HJ, Diefenbach R, Keweloh H (1992) Conversion of cis-unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity. Appl Environ Microbiol 58:1847–1852PubMedGoogle Scholar
  11. Heipieper HJ, Weber FJ, Sikkema J, Keweloh H, de Bont JAM (1994) Mechanisms behind resistance of whole cells to toxic organic solvents. Trends Biotechnol 12:409–415CrossRefGoogle Scholar
  12. Heipieper HJ, Loffeld B, Keweloh H, de Bont JAM (1995) The cis/trans isomerization of unsaturated fatty acids in Pseudomonas putida S12: an indicator for environmental stress due to organic compounds. Chemosphere 30:1041–1051CrossRefGoogle Scholar
  13. Heipieper HJ, Meulenbeld G, van Oirschot Q, de Bont JAM (1996) Effect of environmental factors on the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida S12. Appl Environ Microbiol 62:2773–2777Google Scholar
  14. Heipieper HJ et al (2001) Regiospecific effect of 1-octanol on cistrans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12. Appl Microbiol Biotechnol 57:541–547CrossRefPubMedGoogle Scholar
  15. Heipieper HJ, Meinhardt F, Segura A (2003) The cistrans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism. FEMS Microbiol Lett 229:1–7CrossRefPubMedGoogle Scholar
  16. Holtwick R, Meinhardt F, Keweloh H (1997) cistrans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8. Appl Environ Microbiol 63:4292–4297PubMedGoogle Scholar
  17. Holtwick R, Keweloh H, Meinhardt F (1999) cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type. Appl Environ Microbiol 65:2644–2649Google Scholar
  18. Junker F, Ramos JL (1999) Involvement of the cis/trans isomerase Cti in solvent resistance of Pseudomonas putida DOT-T1E. J Bacteriol 181:5693–5700PubMedGoogle Scholar
  19. Keweloh H, Heipieper HJ (1996) Trans unsaturated fatty acids in bacteria. Lipids 31:129–137PubMedGoogle Scholar
  20. Morita N, Shibahara A, Yamamoto K, Shinkai K, Kajimoto G, Okuyama H (1993) Evidence for cistrans isomerization of a double bond in the fatty acids of the psychrophilic bacterium Vibrio sp. strain ABE-1. J Bacteriol 175:916–918PubMedGoogle Scholar
  21. Morrison WR, Smith LM (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res 5:600–608Google Scholar
  22. Murataliev MB, Klein M, Fulco A, Feyereisen R (1997) Functional interactions in cytochrome P450BM3: flavin semiquinone intermediates, role of NADP(H), and mechanism of electron transfer by the flavoprotein domain. Biochemistry 36:8401–8412CrossRefPubMedGoogle Scholar
  23. Okuyama H, Ueno A, Enari D, Morita N, Kusano T (1998) Purification and characterization of 9-hexadecenoic acid cistrans isomerase from Pseudomonas sp strain E-3. Arch Microbiol 169:29–35CrossRefPubMedGoogle Scholar
  24. O’Leary MH (1980) Determination of heavy-atom isotope effects on enzyme-catalyzed reactions. In: Purich DL (ed) Enzyme kinetics and mechanism. Academic, New York, pp 83–103Google Scholar
  25. O’Neil JR (1986) Theoretical and experimental aspects of isotopic fractionation. In: Valley JW, Taylor HP, O’Neil JR (eds) Stable isotopes in high temperature geological processes. Mineralogical Society of America, Washington, pp 1–37Google Scholar
  26. Pedrotta V, Witholt B (1999) Isolation and characterization of the cistrans-unsaturated fatty acid isomerase of Pseudomonas oleovorans GPo12. J Bacteriol 181:3256–3261PubMedGoogle Scholar
  27. Ramos JL et al (1997) Mechanisms for solvent tolerance in bacteria. J Biol Chem 272:3887–3890CrossRefPubMedGoogle Scholar
  28. Ramos JL et al (2002) Mechanisms of solvent tolerance in gram-negative bacteria. Annu Rev Microbiol 56:743–768CrossRefPubMedGoogle Scholar
  29. Richnow HH, Annweiler E, Michaelis W, Meckenstock RU (2003a) Microbial in situ degradation of aromatic hydrocarbons in a contaminated aquifer monitored by carbon isotope fractionation. J Contam Hydrol 65:101–120CrossRefPubMedGoogle Scholar
  30. Richnow HH, Meckenstock RU, Reitzel LA, Baun A, Ledin A, Christensen TH (2003b) In situ biodegradation determined by carbon isotope fractionation of aromatic hydrocarbons in an anaerobic landfill leachate plume (Vejen, Denmark). J Contam Hydrol 64:59–72CrossRefPubMedGoogle Scholar
  31. von Wallbrunn A, Richnow HH, Neumann G, Meinhardt F, Heipieper HJ (2003) Mechanism of cistrans isomerization of unsaturated fatty acids in Pseudomonas putida. J Bacteriol 185:1730–1733CrossRefPubMedGoogle Scholar
  32. Yoshizawa K, Kagawa Y, Shiota Y (2000a) Kinetic isotope effects in a C–H bond dissociation by the iron-oxo species of cytochrome P450. J Phys Chem B 104:12365–12370CrossRefGoogle Scholar
  33. Yoshizawa K, Shiota Y, Kagawa Y (2000b) Energetics for the oxygen rebound mechanism of alkane hydroxylation by the iron-ore species of cytochrome P450. Bull Chem Soc Jpn 73:2669–2673CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Hermann J. Heipieper
    • 1
  • Grit Neumann
    • 1
  • Nadja Kabelitz
    • 1
  • Matthias Kastner
    • 1
  • Hans Hermann Richnow
    • 1
  1. 1.Department of BioremediationCentre for Environmental Research (UFZ) Leipzig-HalleLeipzigGermany

Personalised recommendations